Human growth hormone (“hGH”) is produced in and released from the pituitary gland. The 22-kDa peptide hormone participates in much of the regulation of normal human growth and development. This pituitary hormone exhibits a multitude of biological effects, including linear growth (somatogenesis), lactation, activation of macrophages, and insulin-like effects, among others. These biological effects are mediated by the interaction between hGH and specific hGH Receptors (hGHR) on the surface of target cells through a cascade of biochemical events (see, for example, Isaksson et al., Annu. Rev. Physiol. 47:483-499, 1985; Friedrichsen et al., Mol. Endocrino. 15:136-148, 2001; Herrington et al., Trends Endocrinol Metab. 12:252-257, 2001).
Recombinant human Growth Hormone (rhGH) is used to treat growth failure due to deficiency of endogenous hGH secretion. Both children and adult patients with hGH deficiency have been treated successfully with rhGH. Other treatment applications for conditions that may involve short stature are in patients for Turner syndrome, chronic renal failure, Prader-Willi syndrome, and idiopathic short stature.
Supplemental hGH is a desirable treatment in cases of growth hormone deficiency for both children and adults. The half-life for the serum clearance of intravenous (i.v.) rhGH in healthy adults is approximately 20 min. The peak serum concentration for subcutaneous (s.c.) rhGH occurs in several hours after injection with an elimination half-life of 3 to 8 h. Treatment with hGH requires s.c. injection three times a week, or once daily to maintain suitable serum levels of hGH. For patients chronically receiving hGH, the frequent injections often result in patient compliance problems. From 1999 to 2004, a sustained-release form of rhGH (Nutropin Depot by Genentech and Alkermes) was in the market, allowing for fewer injections every 2 or 4 weeks instead of daily. The product was withdrawn due to high manufacturing costs. Several versions of PEGylated hGH (see, for example, Cox et al., Endocrinol. 148:1590-1597, 2007; Webster et al., Xenobiotica. 38: 1340-1351, 2008) have been reported to show longer half-life than rhGH yet often at the expense of bioactivities as a result of the PEGylation of the protein. It is therefore desirable to have a long-acting hGH with high activity that can be produced at a reasonable cost.
Immunoglobulins of IgG class are among the most abundant proteins in human blood. Their circulation half-lives can reach as long as 21 days. Fusion proteins have been reported to combine the Fc regions of IgG with the domains of another protein, such as various cytokines and soluble receptors (see, for example, Capon et al., Nature 337:525-531, 1989; Chamow et al., Trends Biotechnol. 14:52-60 (1996); U.S. Pat. Nos. 5,116,964 and 5,541,087). The prototype fusion protein is a homodimeric protein linked through cysteine residues in the hinge region of IgG Fc, resulting in a molecule similar to an IgG molecule without the CH1 domains and light chains. Due to the structural homology, Fc fusion proteins exhibit in vivo pharmacokinetic profile comparable to that of human IgG with a similar isotype. This approach has been applied to several therapeutically important cytokines, such as EPO, and G-CSF and soluble receptors, such as TNF-Rc and IL-5—Rc (see, for example, U.S. Pat. Nos. 5,349,053 and 6,224,867). To extend the circulating half-life of hGH and/or to increase its biological activity, it is desirable to make fusion proteins containing hGH linked to the Fc portion of the human IgG protein as disclosed or described in this invention.
In most of the reported Fc fusion protein molecules, a hinge region serves as a spacer between the Fc region and the cytokine or soluble receptor at the amino-terminus, allowing these two parts of the molecule to function separately (see, for example, Ashkenazi et al., Current Opinion in Immunology, 9:195-200, 1997). It is desirable that a human GH (rHG) fusion protein with an appropriate peptide linker between the hGH and Fc moieties (hGH-L-Fc) may be as good as or more active than rhGH, with in vitro activity similar to, better than, or preferably at least 2-fold as that of rhGH on a molar basis. It is discovered according to this invention that an added peptide linker present between hGH and a human IgG Fc variant may enhance the in vitro biological activity of the hGH-L-Fc molecule in two ways: (1) keeping the Fc region away from the hGHR binding sites on hGH, and (2) keeping one hGH from the other hGH molecule, so both hGH molecules can interact with hGHR on cell surface independently. For the present invention, a flexible peptide linker of about 20 or fewer amino acids in length is preferred. It is also desirable and more preferable to use a peptide linker comprising of two or more of the following amino acids: glycine, serine, alanine, and threonine.
The Fc region of human immunoglobulins plays a significant role in immune defense for the elimination of pathogens. Effector functions of IgG are mediated by the Fc region through two major mechanisms: (1) binding to the cell surface Fc receptors (FcγRs) can lead to ingestion of pathogens by phagocytosis or lysis by killer cells via the antibody-dependent cellular cytotoxicity (ADCC) pathway, or (2) binding to the C1q part of the first complement component C1 initiates the complement-dependent cytotoxicity (CDC) pathway, resulting in the lysis of pathogens. Among the four human IgG isotypes, IgG1 and IgG3 are effective in binding to FcγR. The binding affinity of IgG4 to FcγR is an order of magnitude lower than that of IgG1 or IgG3, while binding of IgG2 to FcγR is below detection. Human IgG1 and IgG3 are also effective in binding to C1q and activating the complement cascade. Human IgG2 fixes complement poorly, and IgG4 appears quite deficient in the ability to activate the complement cascade (see, for example, Jefferis et al., Immunol. Rev. 163:59-76, 1998). For therapeutic use in humans, it is essential that when hGH-L-Fc binds to receptors on the cell surface of target cells, the Fc region of the fusion protein will not mediate undesirable effector functions, leading to the lysis or removal of these cells. Accordingly, the Fc region of hGH-L-Fc must be of a non-lytic nature, i.e. the Fc region must be inert in terms of binding to FcγRs and C1q for the triggering of effector functions. It is clear that none of the naturally occurring IgG isotypes is suitable for use to produce the hGH-L-Fc fusion protein. To obtain a non-lytic Fc, certain amino acids of the natural Fc region have to be mutated for the attenuation of the effector functions.
By comparing amino acid sequences of human and murine IgG isotypes, a portion of Fc near the N-terminal end of the CH2 domain is implicated to play a role in the binding of IgG Fc to FcγRs. The importance of a motif at positions 234 to 237 has been demonstrated using genetically engineered antibodies (see, for example, Duncan et al., Nature 332:563-564, 1988). The numbering of the amino acid residues is according to the EU index as described in Kabat et al. (in Sequences of Proteins of Immunological Interest, 5th Edition, United States Department of Health and Human Services, 1991). Among the four human IgG isotypes, IgG1 and IgG3 bind FcγRs the best and share the sequence Leu234-Leu-Gly-Gly237 (only IgG1 is shown in FIG. 1). In IgG4, which binds FcγRs with a lower affinity, this sequence contains a single amino acid substitution, Phe for Leu at position 234. In IgG2, which does not bind FcγRs, there are two substitutions and a deletion leading to Val234-Ala-Gly237 (FIG. 1). To minimize the binding of Fc to FcγR and hence the ADCC activity, Leu235 in IgG4 has been replaced by Ala (see, for example, Hutchins et al., Proc. Natl. Acad. Sci. USA 92:11980-11984, 1995). IgG1 has been altered in this motif by replacing Glu233-Leu-Leu235 with Pro233-Val-Ala235, which is the sequence from IgG2. This substitution resulted in an IgG1 variant devoid of FcγR-mediated ability to deplete target cells in mice (see, for example, Isaacs et al., J. Immunol. 161: 3862-3869, 1998).
A second portion that appears to be important for both FcγR and C1q binding is located near the carboxyl-terminal end of CH2 domain of human IgG (see, for example, Duncan et al., Nature 332:738-740, 1988). Among the four human IgG isotypes, there is only one site within this portion that shows substitutions: Ser330 and Ser331 in IgG4 replacing Ala330 and Pro331 present in IgG1, IgG2, and IgG3 (FIG. 1). The presence of Ser330 does not affect the binding to FcγR or C1q. The replacement of Pro331 in IgG1 by Ser virtually abolished IgG1 ability to C1q binding, while the replacement of Ser331 by Pro partially restored the complement fixation activity of IgG4 (see, for example, Tao et al., J. Exp. Med. 178:661-667, 1993; Xu et al., J. Biol. Chem. 269:3469-3474, 1994).
We discover that at least three Fc variants (vFc) can be designed for the production of hGH-L-vFc fusion proteins (FIG. 1). Human IgG2 Fc does not bind FcγR but showed weak complement activity. An Fcγ2 variant with Pro331Ser mutation should have less complement activity than natural Fcγ2 while remain nonbonding to FcγR. IgG4 Fc is deficient in activating the complement cascade, and its binding affinity to FcγR is about an order of magnitude lower than that of the most active isotype, IgG1. An Fcγ4 variant with Leu235Ala mutation should exhibit minimal effector functions as compared to the natural Fcγ4. The Fcγ1 variant with Leu234Val, Leu235Ala and Pro331Ser mutations also will exhibit much less effector functions than the natural Fcγ1. These Fc variants are more suitable for the preparation of the hGH fusion proteins than naturally occurring human IgG Fc. It is possible that other replacements can be introduced for the preparation of a non-lytic Fc without compromising the circulating half-life or causing any undesirable conformational changes.
There are many advantages with the present invention. The good activity and/or preferably prolonged presence of the hGH-L-vFc fusion protein in the serum can lead to lower dosages as well as less frequent injections. Less fluctuations of the drug in serum concentrations also means improved safety and tolerability. Less frequent injections may result in better patient compliance and quality of life. The hGH-L-vFc fusion protein containing a non-lytic Fc variant will therefore contribute significantly to the long term management of hGH deficiency in patients that may involve short stature and other conditions including Turner syndrome, chronic renal failure, Prader-Willi syndrome, and idiopathic short stature.